Cereblon

Angiogenesis, in particular anti-angiogenesis, is an area of particular therapeutic interest in cancer treatment. Several anti-angiogenic agents are in the final stages of clinical trials. One of these agents, thalidomide, best known for its teratogenic potential, is showing promise against several tumor types. Thalidomide has been shown previously to require bio-activation to exert its anti-angiogenic effect in isolated blood vessels and endothelial cells. In this work, we confirmed these findings using the in utero chicken embryo chorioallantoic membrane (CAM) system. In particular, the anti-angiogenic effect of thalidomide is significantly enhanced by activation by human but not by rat liver microsomes. We also showed in the CAM assay that hydroxylation of thalidomide at either the 1'- or 5-position retained anti-angiogenic activity whereas its hydroxylation at the 4-position led to an inactive compound. We further demonstrated that thalidomide shows weak anti-proliferative activity against MDA-MB-231 human breast cancer cells in culture. Thalidomide showed slightly more anti-proliferative activity, however, against the SH-SY5Y human neuroblastoma and human umbilical vein endothelial cell (HUVEC) types. Furthermore, incubation of thalidomide with human liver microsomes added no additional anti-proliferative effect in these cell types versus thalidomide given alone. Finally, we report that none of the thalidomide metabolites tested had any anti-proliferative effect against the breast or neuroblastoma cells, but do possess appreciable anti-proliferative activity against the endothelial cells. In summary, this work suggests that hydroxylated thalidomide analogs based on putative metabolites of the drug possess significant anti-angiogenic activity and that exploring further derivatives of these as potential anti-angiogenic agents warrants further merit[1].

The teratogenicity of the chemotherapeutic drug thalidomide is species-specific and affects humans, non-human primates, and rabbits. The primary oxidation of thalidomide in previously investigated rodents predominantly resulted in the formation of deactivated 5'-hydroxythalidomide. In the current study, similar in vivo biotransformations to 5-hydroxythalidomide and 5'-hydroxythalidomide were confirmed by the analysis of blood plasma from male rabbits, a thalidomide-sensitive species, after oral administration of thalidomide (2.0 mg/kg). Similar levels of thalidomide in seminal plasma and in blood plasma were detected using liquid chromatography-tandem mass spectrometry at 4 hr and 7 hr after oral doses in male rabbits. Seminal plasma concentrations of 5-hydroxythalidomide and 5'-hydroxythalidomide were also seen in male rabbits in a roughly similar time-dependent manner to those in the blood plasma after oral doses of thalidomide (2.0 mg/kg). Furthermore, the values generated by a simplified physiologically based pharmacokinetic rabbit model were in agreement with the measured in vivo blood plasma data under metabolic ratios of 0.01 for the hepatic intrinsic clearance of thalidomide to both unconjugated 5-hydroxythalidomide and 5'-hydroxythalidomide. These results suggest that metabolic activation of thalidomide may be dependent on rabbit liver enzymes just it was for cytochrome P450 enzymes in humanized-liver mice; in contrast, rodent livers predominantly mediate biotransformation of thalidomide to 5'-hydroxythalidomide. A developmental toxicity test system with experimental animals that involves intravaginal exposures to the chemotherapeutic drug thalidomide via semen should be considered in the future[2].

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)‐containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide‐ and 5‐hydroxythalidomide‐induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell‐free protein synthesis system, PLZF was identified as a thalidomide‐dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5‐hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5‐hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide‐dependent CRBN substrate SALL4, was induced by thalidomide or 5‐hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide‐induced phenotypes. Our findings implicate PLZF as an important thalidomide‐induced CRBN neosubstrate involved in thalidomide teratogenicity[3].

To examine the 5‐hydroxythalidomide‐dependent degradation of overexpressed PLZF, SALL4 or IKZF1, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐CRBN‐WT and 20 ng pcDNA3.1(+)‐AGIA‐SALL4, pcDNA3.1(+)‐AGIA‐PLZF or pcDNA3.1(+)‐AGIA‐IKZF1. After the cells were transfected for 8 h, they were treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium at the indicated times and concentrations (Fig 4C). For endogenous SALL4, PLZF, IKZF1, HuH7 or THP‐1 cells were cultured in 48‐well plates and treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium at the indicated times and concentrations (Fig 4D and E).[3]

To examine the species specificity of IMiD‐dependent protein degradation, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐(mouse or chicken) CRBN‐WT or CRBN‐IV and 20 ng pcDNA3.1(+)‐AGIA‐(mouse or chicken) PLZF or pcDNA3.1(+)‐AGIA‐(mouse or chicken) SALL4. After the cells were transfected for 8 h, they were treated with thalidomide or DMSO (0.1%) in the culture medium for the times and concentrations indicated in Fig 5A–D.[3]

To examine whether 5‐hydroxythalidomide induced the degradation of mouse or chicken SALL4 or PLZF, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐(mouse or chicken) CRBN‐WT and 20 ng pcDNA3.1(+)‐AGIA‐(mouse or chicken) SALL4 or pcDNA3.1(+)‐AGIA‐(mouse or chicken) PLZF. After the cells were transfected for 8 h, they were treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium for the times and concentrations indicated in Fig 5E–H.[3]

In all experiments, cells were lysed by boiling in 1× sample buffer containing 5% 2‐mercaptoethanol and the lysates were analysed by immunoblotting.

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Quantitative real‐time PCR (qRT–PCR)[3]
To demonstrate decreased post‐translational level of PLZF protein, PLZF mRNA expression in HEK293T, HuH7 or THP‐1 cells treated with DMSO or lenalidomide for 24 h was assessed by qRT–PCR. To analyse SALL4 or PLZF mRNA expression, HuH7 cells treated with DMSO, thalidomide or 5‐hydroxythalidomide for 24 h were assessed by qRT–PCR. Total RNA was isolated from HEK293T, HuH7 or THP‐1 cells treated with DMSO or lenalidomide for 24 h using a SuperPrep cell lysis kit. cDNA was synthesised using a SuperPrep RT kit according to the manufacturer's protocol. RT–PCR was performed using a KOD SYBR qPCR Mix, and data were normalised against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) mRNA levels. PCR primers were as follows: PLZF FW 5′‐GCACAGTTTTCGAAGGAGGA‐3′, PLZF RV 5′‐GGCCATGTCAGTGCCAGT‐3′, SALL4 FW 5′‐GGTCCTCGAGCAGATCTTGT‐3′, SALL4 RV 5′‐GGCATCCAGAGACAGACCTT‐3′, GAPDH FW 5′‐AGCAACAGGGTGGTGGAC‐3′, GAPDH RV 5′‐GTGTGGTGGGGGACTGAG‐3′.

Thalidomide treatment of chicken embryos[3]
A solid crystal of thalidomide was resolved in 45% 2‐hydroxypropyl‐beta‐cyclodextrin for 1–2 h at 60°C to make thalidomide (2 μg/μl, 7.8 mM) or 5‐hydroxythalidomide (2 μg/μl, 7.3 mM) stock solution. This stock was mixed with the same volume of 2× Hanks buffer as a working solution (1 μg/μl, 3.9 mM thalidomide or 3.6 mM 5‐hydroxythalidomide). To apply thalidomide to an embryo, a small hole was opened at the amnion above the right forelimb bud at HH st. 18 (E3), and the working solution was injected in a space between the amnion and right forelimb bud. In the case of samples for in situ hybridisation, to induce a strong effect on target proteins of thalidomide or 5‐hydroxythalidomide for short exposure time, immunofluorescence and immunoblot analyses, embryos were treated with 30 μl of working solution and incubated until HH st. 22/23 (E4). In the case of samples of skeletal pattern analysis, to reduce lethality and increase the collection rate of chicken embryos showing teratogenic phenotypes in limb bud at HH st. 36 (E10), embryos were treated three times with 10 μl of working solution every 12 h and incubated until approximately HH st. 36 (E10).

[1]. Effects of Putative Hydroxylated Thalidomide Metabolites on Blood Vessel Density in the Chorioallantoic Membrane (CAM) Assay and on Tumor and Endothelial Cell Proliferation. Biol Pharm Bull. 2002 May;25(5):597-604.

5-Hydroxythalidomide is a member of phthalimides.

C13H10N2O5

274.2289

274.059

C, 56.94; H, 3.68; N, 10.22; O, 29.17

64567-60-8

Thalidomide-4-OH;5054-59-1

5743568

Gray to brown solid powder

1.611g/cm3

600ºC at 760mmHg

316.7ºC

1.679

0.007

2

5

1

20

503

0

O=C1CCC(N2C(=O)C3C=CC(=CC=3C2=O)O)C(=O)N1

LJBQRRQTZUJWRC-UHFFFAOYSA-N

InChI=1S/C13H10N2O5/c16-6-1-2-7-8(5-6)13(20)15(12(7)19)9-3-4-10(17)14-11(9)18/h1-2,5,9,16H,3-4H2,(H,14,17,18)

2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindole-1,3-dione

5-hydroxythalidomide; 64567-60-8; 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione; Thalidomide-5-OH; 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindole-1,3-dione; (+/-)-Hydroxythalidomide; CHEMBL182442; 29V976C3CJ;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~62.5 mg/mL (~227.91 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)